Partners in crime: Tbf1 and Vid22 promote expansions of long human telomeric repeats at an interstitial chromosome position in yeast

Abstract

In humans, telomeric repeats (TTAGGG)n are known to be present at internal chromosomal sites. These interstitial telomeric sequences (ITSs) are an important source of genomic instability, including repeat length polymorphism, but the molecular mechanisms responsible for this instability remain to be understood. Here, we studied the mechanisms responsible for expansions of human telomeric (Htel) repeats that were artificially inserted inside a yeast chromosome. We found that Htel repeats in an interstitial chromosome position are prone to expansions. The propensity of Htel repeats to expand depends on the presence of a complex of two yeast proteins: Tbf1 and Vid22. These two proteins are physically bound to an interstitial Htel repeat, and together they slow replication fork progression through it. We propose that slow progression of the replication fork through the protein complex formed by the Tbf1 and Vid22 partners at the Htel repeat cause DNA strand slippage, ultimately resulting in repeat expansions.

The telomerase reverse transcriptase elongates reversed replication forks at telomeric repeats

“…The experiments with ectopic repeats also indicate that reversed forks can occur in the absence of a t-loop structure. Other potential obstacles to telomere replication include the presence of damage-induced i-loops, or tightly bound shelterin subcomplexes that need to be removed at the passage of the replication fork (12)(13)(14)(15)18). Other intrinsic features of stretches of repetitive DNA, like the abundance of local homology, could also contribute to replication fork reversal at telomeres (16,23).…”

“…The severe telomere replication defects observed in shelterin mutants, from yeast to mammals, suggest that shelterin plays an essential function in assisting semiconservative replication of telomeric repeats (4,8,9). Several telomeric features have been invoked as potential obstacles to fork progression, including the presence of tightly bound proteins, ongoing transcription, or secondary structures like G4-DNA, t-loops, R-loops, and damage-induced i-loops (6,(10)(11)(12)(13)(14)(15). However, the relative contribution of each of these factors to telomere replication stress is not clear.…”

Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures

“…Plasmid construction for replication analysis in yeast: Primers JH92 and JH93 were used to amplify (A 2 G 3 ) 60 from pJH7 to add BsrGI restriction sites to the ends of this PCR product and insert this product into plasmid pRS425 - UIRLB ( 24 ). Clones identified as correct using (i) PCRs flanking the ligation integration site and (ii) Repeat PCR protocol Taq 1 ( Supplementary Table S3 ) were sent for sequencing and the correct clones were named pJH1 (purine lagging strand template) and pJH26 (pyrimidine lagging strand template).…”

“…Yeast cells: Following the methods outlined in ( 24 ), strains containing the yeast two-dimensional gel plasmid were grown, yeast replication intermediates were extracted, digested with restriction enzymes, run on 2D gel and analyzed.…”

“…Two-dimensional gel electrophoresis quantification was carried out as described in ( 24 ) and demonstrated visually in Supplementary Figure S7 . For each biological replicate, of which there were three for yeast and three for human cell replication, the following was performed.…”